PMID:11736651
| Citation |
Han, Q, Fang, J and Li, J (2001) Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase. Biochem. J. 360:617-23 |
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| Abstract |
The present study describes the isolation of a protein from Escherichia coli possessing kynurenine aminotransferase (KAT) activity and its identification as aspartate aminotransferase (AspAT). KAT catalyses the transamination of kynurenine and 3-hydroxykynurenine to kynurenic acid and xanthurenic acid respectively, and the enzyme activity can be easily detected in E. coli cells. Separation of the E. coli protein possessing KAT activity through various chromatographic steps led to the isolation of the enzyme. N-terminal sequencing of the purified protein determined its first 10 N-terminal amino acid residues, which were identical with those of the E. coli AspAT. Recombinant AspAT (R-AspAT), homologously expressed in an E. coli/pET22b expression system, was capable of catalysing the transamination of both l-kynurenine (K(m)=3 mM; V(max)=7.9 micromol.min(-1).mg(-1)) and 3-hydroxy-dl-kynurenine (K(m)=3.7 mM; V(max)=1.25 micromol.min(-1).mg(-1)) in the presence of pyruvate as an amino acceptor, and exhibited its maximum activity at temperatures between 50-60 degrees C and at a pH of approx. 7.0. Like mammalian KATs, R-AspAT also displayed high glutamine transaminase K activity when l-phenylalanine was used as an amino donor (K(m)=8 mM; V(max)=20.6 micromol.min(-1).mg(-1)). The exact match of the first ten N-terminal amino acid residues of the KAT-active protein with that of AspAT, in conjunction with the high KAT activity of R-AspAT, provides convincing evidence that the identity of the E. coli protein is AspAT. |
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| Keywords |
Aspartate Aminotransferases/isolation & purification; Aspartate Aminotransferases/metabolism; Escherichia coli/enzymology; Hydrogen-Ion Concentration; Kinetics; Lyases/isolation & purification; Lyases/metabolism; Substrate Specificity; Thermodynamics; Transaminases/isolation & purification; Transaminases/metabolism |
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